Many halogenated organic contaminants (HOCs) are considered endocrine disruptors and affect the hypothalamic–pituitary–thyroid axis, often by interfering with circulating levels of thyroid hormones (THs). We investigated one potential mechanism for TH disruption, inhibition of sulfotransferase activity. One of the primary roles of TH sulfation is to support the regulation of biologically active T3 through the formation of inactive THs. We investigated TH sulfotransferase inhibition by 14 hydroxylated polybrominated diphenyl ethers (OH BDEs), BDE 47, triclosan, and fluorinated, chlorinated, brominated, and iodinated analogues of 2,4,6-trihalogenated phenol and bisphenol A (BPA). A new mass spectrometry-based method was also developed to measure the formation rates of 3,3′-T2 sulfate (3,3′-T2S). Using pooled human liver cytosol, we investigated the influence of these HOCs on the sulfation of 3,3′-T2, a major substrate for TH sulfation. For the formation of 3,3′-T2S, the Michaelis constant (K m) was 1070 ± 120 nM and the V max was 153 ± 6.6 pmol min–1 (mg of protein)−1. All chemicals investigated inhibited sulfotransferase activity with the exception of BDE 47. The 2,4,6-trihalogenated phenols were the most potent inhibitors followed by the OH BDEs and then halogenated BPAs. The IC50 values for the OH BDEs were primarily in the low nanomolar range, which may be environmentally relevant. In silico molecular modeling techniques were also used to simulate the binding of OH BDE to SULT1A1. This study suggests that some HOCs, including antimicrobial chemicals and metabolites of flame retardants, may interfere with TH regulation through inhibition of sulfotransferase activity.